Abstract

The global emergence of multidrug-resistant tuberculosis has highlighted the need for the development of rapid tests to identify resistance to second-line antituberculosis drugs. Resistance to fluoroquinolones and aminoglycosides develops through nonsynonymous single nucleotide polymorphisms in the gyrA and gyrB genes and the rrs gene, respectively. Using DNA sequencing as the gold standard for the detection of mutations conferring resistance, in conjunction with spoligotyping, we demonstrated heteroresistance in 25% and 16.3% of Mycobacterium tuberculosis isolates resistant to ofloxacin and amikacin, respectively. Characterization of follow-up isolates from the same patients showed that the population structure of clones may change during treatment, suggesting different phases in the emergence of resistance. The presence of underlying mutant clones was identified in isolates which failed to show a correlation between phenotypic resistance and mutation in the gyrA or rrs gene. These clones harbored previously described mutations in either the gyrA or rrs gene, suggesting that rare mutations conferring resistance to ofloxacin or amikacin may not be as important as was previously thought. We concluded that the absence of a correlation between genotypic and phenotypic resistance implies an early phase in the emergence of resistance within the patient. Thus, the diagnostic utility of genetics-based drug susceptibility tests will depend on the proportion of patients whose bacilli are in the process of acquiring resistance in the study setting. These data have implications for the interpretation of molecular and microbiological diagnostic tests for patients with drug-susceptible and drug-resistant tuberculosis who fail to respond to treatment and for those with discordant results.

DNA sequence chromatograms of the QRDR for follow-up isolates from three patients in whom fluoroquinolone resistance emerged. The sequence chromatograms for patient 1 show the emergence of two QRDR variants (D94G and D94A), followed by the selection of the D94G variant. The sequence chromatograms for patient 2 show the presence of at least 3 QRDR variants (D94G, D94S, and D94N), including the “wild-type” (WT) sequence, followed by the selection of the D94N variant. The sequence chromatograms for patient 3 show the persistence of three QRDR variants (WT, A90V, and D94G) for at least 16 months, followed by the selection of the D94G variant.